锌指蛋白C2H2-71通过抑制LIN5来调控番茄可溶性固形物含量

doi:10.1016/j.jia.2024.11.027

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (6): 2190-2202.DOI: 10.1016/j.jia.2024.11.027

锌指蛋白C2H2-71通过抑制LIN5来调控番茄可溶性固形物含量

收稿日期:2024-01-22 修回日期:2024-11-13 接受日期:2024-06-11 出版日期:2025-06-20 发布日期:2025-05-12

ZF protein C2H2-71 regulates the soluble solids content in tomato by inhibiting LIN5

Fangman Li¹, Junshen Lin¹, John Kojo Ahiakpa¹, Wenxian Gai¹, Jinbao Tao¹, Pingfei Ge¹, Xingyu Zhang¹, Yizhuo Mu⁵, Jie Ye¹, Yuyang Zhang^{1, 2, 3, 4#}

¹National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University, Wuhan 430070, China
²Hubei Hongshan Laboratory, Wuhan 430070, China
³Shenzhen Institute of Nutrition and Health, Huazhong Agricultural University, Wuhan 430070, China
⁴Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture/Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518000, China
⁵College of Agricultural and Environmental Science, University of California, Davis, California 95616, USA

Received:2024-01-22 Revised:2024-11-13 Accepted:2024-06-11 Online:2025-06-20 Published:2025-05-12
About author:Fangman Li, E-mail: lfm@mail.hzau.edu.cn; #Correspondence Yuyang Zhang, Tel: +86-27-87282010, E-mail: yyzhang@mail.hzau.edu.cn
Supported by:
This work was supported by the grants from the National Key Research & Development Plan of China (2022YFF1003002; 2022YFD1200502), the National Natural Science Foundation of China (32372696), the Wuhan Biological Breeding Major Project, China (2022021302024852), the HZAU–AGIS Cooperation Fund, China (SZYJY2023022), the Funds for High Quality Development of Hubei Seed Industry, China (HBZY2023B004), the Hubei Agriculture Research System, China (2024HBSTX4-06), and the Funds of National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, China (Horti-3Y-2024-008).

摘要/Abstract

摘要：

可溶性固形物含量(SSC)是番茄果实风味的重要影响因素。研究表明，果实可溶性固形物积累在糖代谢过程中受到转录调控。之前对于C2H2型锌指蛋白的研究主要集中在生长发育方向，但C2H2锌指蛋白对果实可溶性固形物积累的调控机制尚不清楚。本研究选取了8种不同可溶性固形物含量的番茄材料，通过对红熟果实转录组数据中SlC2H2家族基因表达分析，发现SlC2H2-71基因在高SSC材料中的表达量明显低于低SSC材料。利用CRISPR-Cas9系统构建SlC2H2-71敲除系，发现转基因材料红熟果实中可溶性固形物、果糖、葡萄糖、苹果酸和柠檬酸的含量上升，而蔗糖含量却下降。转录组分析表明，突变体果实中糖酸代谢通路相关基因表达被调控，其中细胞壁酸性转化酶SlLIN5表达上调，并通过实时荧光定量PCR证实了这一点。酵母单杂交（Y1H）、35S::UAS-GUS报告系统、双荧光素酶报告系统和凝胶迁移实验（EMSA）表明，SlC2H2-71锌指蛋白可直接结合SlLIN5启动子并抑制其表达。酶活实验表明SlC2H2-71敲除系果实中的细胞壁酸性转化酶活性显著高于对照系。综上所述，SlC2H2-71锌指蛋白可通过抑制细胞壁酸性转化酶SlLIN5基因的表达和其编码的细胞壁酸性转化酶活性来影响番茄红熟果实可溶性固形物积累。

Abstract:

Soluble solids content (SSC) plays an important role in determining the flavor of tomato fruits. Tomato fruit SSC is transcriptionally regulated via sugar metabolism. Previous studies have predominantly focused on the role of C2H2-type zinc finger proteins in tomato growth and development. However, the specific regulatory mechanisms of C2H2 in the accumulation of soluble solids in tomato fruits are not fully understood. This study used eight tomato accessions with varying levels of SSC to study the expression of SlC2H2 family genes in red ripe fruits. The levels of SlC2H2-71 expression were found to be significantly reduced in high-SSC accessions compared to low-SSC accessions. Several Slc2h2-71 mutant lines were developed using the CRISPR-Cas9 system, which led to elevated levels of soluble solids, fructose, glucose, malic acid, and citric acid in mature red ripe fruits. However, the sucrose content in the edited Slc2h2-71 mutant lines was generally lower. RNA-seq analysis revealed that fruits from the mutant lines had altered expression of genes related to the sugar and acid metabolic pathways, which was further confirmed by quantitative real-time PCR. Specifically, the expression of SlLIN5 encoding the cell wall invertase (CWIN) was elevated. The yeast one-hybrid (Y1H) assay, 35S::UAS-GUS, dual-luciferase reporter systems and electrophoretic mobility shift assay (EMSA) demonstrated that C2H2-71 regulates tomato sugar metabolism by directly binding to the promoter region of SlLIN5, culminating in the repression of its transcriptional activity. The activity of acid invertase exhibited a significantly higher level in the SlC2H2-71 knock-out lines compared to the control lines. In summary, the regulation of tomato fruit SSC by C2H2-71 involves the inhibition of SlLIN5 expression.

Key words: Solanum lycopersicum L. , C2H2 zinc finger protein , transcription factor , fruit flavor , cell wall invertase , LIN5

Fangman Li, Junshen Lin, John Kojo Ahiakpa, Wenxian Gai, Jinbao Tao, Pingfei Ge, Xingyu Zhang, Yizhuo Mu, Jie Ye, Yuyang Zhang. 锌指蛋白C2H2-71通过抑制LIN5来调控番茄可溶性固形物含量[J]. Journal of Integrative Agriculture, 2025, 24(6): 2190-2202.

Fangman Li, Junshen Lin, John Kojo Ahiakpa, Wenxian Gai, Jinbao Tao, Pingfei Ge, Xingyu Zhang, Yizhuo Mu, Jie Ye, Yuyang Zhang. ZF protein C2H2-71 regulates the soluble solids content in tomato by inhibiting LIN5[J]. Journal of Integrative Agriculture, 2025, 24(6): 2190-2202.

参考文献

Aprea E, Charles M, Endrizzi I, Laura Corollaro M, Betta E, Biasioli F, Gasperi F. 2017. Sweet taste in apple: The role of sorbitol, individual sugars, organic acids and volatile compounds. Scientific Reports, 7, 44950.

Baxter C, Carrari F, Bauke A, Overy S, Hill S, Quick P, Fernie A, Sweetlove L. 2005. Fruit carbohydrate metabolism in an introgression line of tomato (Lycopersicon esculentum) with increased fruit soluble solids. Plant and Cell Physiology, 46, 425–437.

Berg J M, Shi Y. 1996. The galvanization of biology: A growing appreciation for the roles of zinc. Science, 271, 1081–1085.

Chen C, Chen H, Zhang Y, Thomas H R, Frank M H, He Y, Xia R. 2020. Tbtools: An integrative toolkit developed for interactive analyses of big biological data. Molecular Plant, 13, 1194–1202.

Cheng H, Kong W, Tang T, Ren K, Zhang K, Wei H, Lin T. 2022. Identification of key gene networks controlling soluble sugar and organic acid metabolism during oriental melon fruit development by integrated analysis of metabolic and transcriptomic analyses. Frontiers in Plant Science, 13, 830517.

Colantonio V, Ferrao L, Tieman D M, Bliznyuk N, Sims C, Klee H J, Munoz P, Resende M J. 2022. Metabolomic selection for enhanced fruit flavor. Proceedings of the National Academy of Sciences of the United States of America, 119, e2115865119.

Dobin A, Davis C A, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras T R. 2013. Star: Ultrafast universal RNA-seq aligner. Bioinformatics, 29, 15–21.

Duan Y, Yang L, Zhu H, Zhou J, Sun H, Gong H. 2021. Structure and expression analysis of sucrose phosphate synthase, sucrose synthase and invertase gene families in Solanum lycopersicum. International Journal of Molecular Sciences, 22, 4698.

Eshed Y, Zamir D. 1995. An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated QTL. Genetics, 141, 1147–1162.

Fan Z, Hasing T, Johnson T S, Garner D M, Barbey C R, Colquhoun T A, Sims C A, Resende M F R, Whitaker V M. 2021. Strawberry sweetness and consumer preference are enhanced by specific volatile compounds. Horticulture Research, 8, 66.

Fang H, Shi Y, Liu S, Jin R, Sun J, Grierson D, Li S, Chen K. 2023. The transcription factor CitZAT5 modifies sugar accumulation and hexose proportion in citrus fruit. Plant Physiology, 192, 1858–1876.

Guan Y, Hwarari D, Korboe H M, Ahmad B, Cao Y, Movahedi A, Yang L. 2023. Low temperature stress-induced perception and molecular signaling pathways in plants. Environmental and Experimental Botany, 207, 105190.

Han G, Lu C, Guo J, Qiao Z, Sui N, Qiu N, Wang B. 2020. C2H2 zinc finger proteins: Master regulators of abiotic stress responses in plants. Frontiers in Plant Science, 11, 115.

Hu X, Zhu L, Zhang Y, Xu L, Li N, Zhang X, Pan Y. 2019. Genome-wide identification of C2H2 zinc-finger genes and their expression patterns under heat stress in tomato (Solanum lycopersicum L.). PeerJ, 7, e7929.

Huang T, Yu D, Wang X. 2021. VvWRKY22 transcription factor interacts with VvSnRK1.1/VvSnRK1.2 and regulates sugar accumulation in grape. Biochemical and Biophysical Research Communications, 554, 193–198.

Hussain A, Liu J, Mohan B, Burhan A, Nasim Z, Bano R, Ameen A, Zaynab M, Mukhtar M S, Pajerowska-Mukhtar K M. 2022. A genome-wide comparative evolutionary analysis of zinc finger-bed transcription factor genes in land plants. Scientific Reports, 12, 12328.

Jin Y, Ni D A, Ruan Y L. 2009. Posttranslational elevation of cell wall invertase activity by silencing its inhibitor in tomato delays leaf senescence and increases seed weight and fruit hexose level. Plant Cell, 21, 2072–2089.

Kawaguchi K, Takei-Hoshi R, Yoshikawa I, Nishida K, Kobayashi M, Kusano M, Lu Y, Ariizumi T, Ezura H, Otagaki S, Matsumoto S, Shiratake K. 2021. Functional disruption of cell wall invertase inhibitor by genome editing increases sugar content of tomato fruit without decrease fruit weight. Scientific Reports, 11, 21534.

Klee H J, Tieman D M. 2018. The genetics of fruit flavour preferences. Nature Reviews Genetics, 19, 347–356.

Kundu A, Das S, Basu S, Kobayashi Y, Koyama H, Ganesan M, Whelan J. 2019. GhSTOP1, C2H2 type zinc finger transcription factor is essential for aluminum and proton stress tolerance and lateral root initiation in cotton. Plant Biology, 21, 35–44.

Li R, Wang X, Zhang S, Liu X, Zhou Z, Liu Z, Wang K, Tian Y, Wang H, Zhang Y, Cui X. 2021. Two zinc-finger proteins control the initiation and elongation of long stalk trichomes in tomato. Journal of Genetics and Genomics, 48, 1057–1069.

Li X, Tieman D, Alseekh S, Fernie A R, Klee H J. 2023. Natural variations in the Sl-AKR9 aldo/keto reductase gene impact fruit flavor volatile and sugar contents. Plant Journal, 115, 1134–1150.

Liang Y, Bai J, Xie Z, Lian Z, Guo J, Zhao F, Liang Y, Huo H, Gong H. 2023. Tomato sucrose transporter SlSUT4 participates in flowering regulation by modulating gibberellin biosynthesis. Plant Physiology, 192, 1080–1098.

Liao Y, Smyth G K, Shi W. 2014. Featurecounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 30, 923–930.

Liu Y, Offler C E, Ruan Y. 2016. Cell wall invertase promotes fruit set under heat stress by suppressing ROS-independent cell death. Plant Physiology, 1, 163–180.

Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods, 25, 402–408.

Miller J, Mclachlan A D, Klug A. 1985. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. The Embo Journal, 4, 1609–1614.

Robinson M D, Mccarthy D J, Smyth G K. 2010. Edger: A bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 26, 139–140.

Ruan Y L, Jin Y, Yang Y J, Li G J, Boyer J S. 2010. Sugar input, metabolism, and signaling mediated by invertase: Roles in development, yield potential, and response to drought and heat. Molecular Plant, 3, 942–955.

Shang L, Song J, Yu H, Wang X, Yu C, Wang Y, Li F, Lu Y, Wang T, Ouyang B, Zhang J, Larkin R M, Ye Z, Zhang Y. 2021. A mutation in a C2H2-type zinc finger transcription factor contributed to the transition toward self-pollination in cultivated tomato. Plant Cell, 33, 3293–3308.

Stein O, Granot D. 2019. An overview of sucrose synthases in plants. Frontiers in Plant Science, 10, 95.

Tadeo F R, Terol J, Rodrigo M J, Licciardello C, Sadka A. 2020. Fruit growth and development. In: Talon M, Caruso M, Gmitter F G, eds., The Genus Citrus. Woodhead Publishing. pp. 245–269.

Takatsuji H, Mori M, Benfey P N, Ren L, Chua N H. 1992. Characterization of a zinc finger DNA-binding protein expressed specifically in petunia petals and seedlings. The Embo Journal, 11, 241–249.

Tao H, Sun H, Wang Y, Wang X, Guo Y. 2023. Effects of water stress on quality and sugar metabolism in ‘ gala’ apple fruit. Horticultural Plant Journal, 9, 60–72.

Umer M J, Bin Safdar L, Gebremeskel H, Zhao S, Yuan P, Zhu H, Kaseb M O, Anees M, Lu X, He N, Gong C, Liu W. 2020. Identification of key gene networks controlling organic acid and sugar metabolism during watermelon fruit development by integrating metabolic phenotypes and gene expression profiles. Horticulture Research, 7, 193.

Wang C, Wang G, Wen X, Qu X, Zhang Y, Zhang X, Deng P, Chen C, Ji W, Zhang H. 2023. Characteristics and expression analysis of invertase gene family in common wheat (Triticum aestivum L. ). Genes, 14, 41.

Wang R, Shu P, Zhang C, Zhang J, Chen Y, Zhang Y, Du K, Xie Y, Li M, Ma T, Zhang Y, Li Z, Grierson D, Pirrello J, Chen K, Bouzayen M, Zhang B, Liu M. 2022. Integrative analyses of metabolome and genome‐wide transcriptome reveal the regulatory network governing flavor formation in kiwifruit (Actinidia chinensis). New Phytologist, 233, 373–389.

Wang Y, Wang L, Zou Y, Chen L, Cai Z, Zhang S, Zhao F, Tian Y, Jiang Q, Ferguson B J, Gresshoff P M, Li X. 2014. Soybean miR172c targets the repressive AP2 transcription factor NNC1 to activate ENOD40 expression and regulate nodule initiation. Plant Cell, 26, 4782–4801.

Wang Z, Wei X, Yang J, Li H, Ma B, Zhang K, Zhang Y, Cheng L, Ma F, Li M. 2020. Heterologous expression of the apple hexose transporter MdHT2.2 altered sugar concentration with increasing cell wall invertase activity in tomato fruit. Plant Biotechnology Journal, 18, 540–552.

Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, Feng T, Zhou L, Tang W, Zhan L, Fu X, Liu S, Bo X, Yu G. 2021. Clusterprofiler 4.0: A universal enrichment tool for interpreting omics data. Innovation, 2, 100141.

Xie K, Zhang J, Yang Y. 2014. Genome-wide prediction of highly specific guide rna spacers for CRISPR-Cas9-mediated genome editing in model plants and major crops. Molecular Plant, 7, 923–926.

Xiong Y, Yan P, Du K, Li M, Xie Y, Gao P. 2020. Nutritional component analyses of kiwifruit in different development stages by metabolomic and transcriptomic approaches. Journal of the Science of Food and Agriculture, 100, 2399–2409.

Yang J, Zhu L, Cui W, Zhang C, Li D, Ma B, Cheng L, Ruan Y, Ma F, Li M. 2018. Increased activity of MdFRK2, a high-affinity fructokinase, leads to upregulation of sorbitol metabolism and downregulation of sucrose metabolism in apple leaves. Horticulture Research, 5, 12–71.

Yang J, Zhan R, Wang L, Li J, Ma B, Ma F, Li M. 2023. Overexpression of MdFRK2 enhances apple drought resistance by promoting carbohydrate metabolism and root growth under drought stress. Horticultural Plant Journal, 9, 884–897.

Zanor M I, Osorio S, Nunes-Nesi A, Carrari F, Lohse M, Usadel B, Kuhn C, Bleiss W, Giavalisco P, Willmitzer L, Sulpice R, Zhou Y H, Fernie A R. 2009. RNA interference of LIN5 in tomato confirms its role in controlling Brix content, uncovers the influence of sugars on the levels of fruit hormones, and demonstrates the importance of sucrose cleavage for normal fruit development and fertility. Plant Physiology, 150, 1204–1218.

Zhang J, Wu Z, Hu F, Liu L, Huang X, Zhao J, Wang H. 2018. Aberrant seed development in Litchi chinensis is associated with the impaired expression of cell wall invertase genes. Horticulture Research, 5, 39.

Zhang Z, Xing Y, Ramakrishnan M, Chen C, Xie F, Hua Q, Chen J, Zhang R, Zhao J, Hu G, Qin Y. 2022. Transcriptomics-based identification and characterization of genes related to sugar metabolism in ‘hongshuijing’ pitaya. Horticultural Plant Journal, 8, 450–460.

Zheng F, Cui L, Li C, Xie Q, Ai G, Wang J, Yu H, Wang T, Zhang J, Ye Z, Yang C. 2022. Hair interacts with SlZFP8-like to regulate the initiation and elongation of trichomes by modulating SlZFP6 expression in tomato. Journal of Experimental Botany, 73, 228–244.

Zhou Z, Yuan Y, Wang K, Wang H, Huang J, Yu H, Cui X. 2022. Rootstock–scion interactions affect fruit flavor in grafted tomato. Horticultural Plant Journal, 8, 499–510.

锌指蛋白C2H2-71通过抑制LIN5来调控番茄可溶性固形物含量

ZF protein C2H2-71 regulates the soluble solids content in tomato by inhibiting LIN5

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